From the Cover: Structural basis for the glucan phosphatase activity of Starch Excess4

Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydrate-binding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.     

From the Cover: Generic theory for channel sinuosity

Sinuous patterns traced by fluid flows are a ubiquitous feature of physical landscapes on Earth, Mars, the volcanic floodplains of the Moon and Venus, and other planetary bodies. Typically discussed as a consequence of migration processes in meandering rivers, sinuosity is also expressed in channel types that show little or no indication of meandering. Sinuosity is sometimes described as “inherited” from a preexisting morphology, which still does not explain where the inherited sinuosity came from. For a phenomenon so universal as sinuosity, existing models of channelized flows do not explain the occurrence of sinuosity in the full variety of settings in which it manifests, or how sinuosity may originate. Here we present a generic theory for sinuous flow patterns in landscapes. Using observations from nature and a numerical model of flow routing, we propose that flow resistance (representing landscape roughness attributable to topography or vegetation density) relative to surface slope exerts a fundamental control on channel sinuosity that is effectively independent of internal flow dynamics. Resistance-dominated surfaces produce channels with higher sinuosity than those of slope-dominated surfaces because increased resistance impedes downslope flow. Not limited to rivers, the hypothesis we explore pertains to sinuosity as a geomorphic pattern. The explanation we propose is inclusive enough to account for a wide variety of sinuous channel types in nature, and can serve as an analytical tool for determining the sinuosity a landscape might support.     

Structural basis for antagonism and resistance of bicalutamide in prostate cancer

Carcinoma of the prostate is the most commonly diagnosed cancer in men. The current pharmacological treatment of choice for progressive androgen-dependent prostate cancer is the nonsteroidal antiandrogen, bicalutamide, either as monotherapy or with adjuvant castration or luteinizing hormone-releasing hormone superagonists to block the synthesis of endogenous testosterone. To date, no nonsteroidal or antagonist-bound androgen receptor (AR) structure is available. We solved the x-ray crystal structure of the mutant W741L AR ligand-binding domain bound to R-bicalutamide at 1.8-A resolution. This mutation confers agonist activity to bicalutamide and is likely involved in bicalutamide withdrawal syndrome. The three-dimensional structure demonstrates that the B ring of R-bicalutamide in the W741L mutant is accommodated at the location of the indole ring of Trp-741 in the WT AR bound to dihydrotestosterone. Knowledge of the binding mechanism for R-bicalutamide will provide molecular rationale for the development of new antiandrogens and selective AR modulators.     

Structural basis for agonism and antagonism of hepatocyte growth factor

Hepatocyte growth factor (HGF) is an activating ligand of the Met receptor tyrosine kinase, whose activity is essential for normal tissue development and organ regeneration but abnormal activation of Met has been implicated in growth, invasion, and metastasis of many types of solid tumors. HGF has two natural splice variants, NK1 and NK2, which contain the N-terminal domain (N) and the first kringle (K1) or the first two kringle domains of HGF. NK1, which is a Met agonist, forms a head-to-tail dimer complex in crystal structures and mutations in the NK1 dimer interface convert NK1 to a Met antagonist. In contrast, NK2 is a Met antagonist, capable of inhibiting HGF’s activity in cell proliferation without clear mechanism. Here we report the crystal structure of NK2, which forms a “closed” monomeric conformation through interdomain interactions between the N- domain and the second kringle domain (K2). Mutations that were designed to open up the NK2 closed conformation by disrupting the N/K2 interface convert NK2 from a Met antagonist to an agonist. Remarkably, this mutated NK2 agonist can be converted back to an antagonist by a mutation that disrupts the NK1/NK1 dimer interface. These results reveal the molecular determinants that regulate the agonist/antagonist properties of HGF NK2 and provide critical insights into the dimerization mechanism that regulates the Met receptor activation by HGF.     

From the Cover: Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH₂]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.     

From the Cover: Peptide antagonism as a mechanism for NK cell activation

Inhibition of natural killer (NK) cells is mediated by MHC class I receptors including the killer cell Ig-like receptor (KIR). We demonstrate that HLA-C binding peptides can function as altered peptide ligands for KIR and antagonize the inhibition mediated by KIR2DL2/KIR2DL3. Antagonistic peptides promote clustering of KIR at the interface of effector and target cells, but do not result in inhibition of NK cells. Our data show that, as for T cells, small changes in the peptide content of MHC class I can regulate NK cell activity.     

From the Cover: Neural basis of global resting-state fMRI activity

Functional MRI (fMRI) has uncovered widespread hemodynamic fluctuations in the brain during rest. Recent electroencephalographic work in humans and microelectrode recordings in anesthetized monkeys have shown this activity to be correlated with slow changes in neural activity. Here we report that the spontaneous fluctuations in the local field potential (LFP) measured from a single cortical site in monkeys at rest exhibit widespread, positive correlations with fMRI signals over nearly the entire cerebral cortex. This correlation was especially consistent in a band of upper gamma-range frequencies (40–80 Hz), for which the hemodynamic signal lagged the neural signal by 6–8 s. A strong, positive correlation was also observed in a band of lower frequencies (2–15 Hz), albeit with a lag closer to zero. The global pattern of correlation with spontaneous fMRI fluctuations was similar whether the LFP signal was measured in occipital, parietal, or frontal electrodes. This coupling was, however, dependent on the monkey''s behavioral state, being stronger and anticipatory when the animals’ eyes were closed. These results indicate that the often discarded global component of fMRI fluctuations measured during the resting state is tightly coupled with underlying neural activity.     

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6–8 Å outward through a narrow groove formed by the S1, S2, and S3 segments, rotates ∼30°, and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 3₁₀-helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4–S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.Voltage-gated sodium (Na_V) channels are responsible for initiation and propagation of action potentials in nerve, muscle, and endocrine cells (1, 2). They are members of the structurally homologous superfamily of voltage-gated ion channel proteins that also includes voltage-gated potassium (K_V), voltage-gated calcium (Ca_V), and cyclic nucleotide-gated (CNG) channels (3). Mammalian Na_V and Ca_V channels consist of four homologous domains (I through IV), each containing six transmembrane segments (S1 through S6) and a membrane-reentrant pore loop between the S5 and S6 segments (1, 3). Segments S1–S4 of the channel form the voltage-sensing domain (VSD), and segments S5 and S6 and the membrane-reentrant pore loop form the pore. The bacterial Na_V channel NaChBac and its relatives consist of tetramers of four identical subunits, which closely resemble one domain of vertebrate Na_V and Ca_V channels, but provide much simpler structures for studying the mechanism of voltage sensing (4, 5). The hallmark feature of the voltage-gated ion channels is the steep voltage dependence of activation, which derives from the voltage-driven outward movement of gating charges in response to the membrane depolarization (6, 7). The S4 transmembrane segment in the VSD has four to seven arginine residues spaced at 3-aa intervals, which serve as gating charges in the voltage-sensing mechanism (8–15). The intracellular S4–S5 linker that connects the VSD to the pore plays a key role in coupling voltage-dependent conformational changes in the VSD to opening and closing of the pore (16). The gating charges are pulled in by the internally negative transmembrane electric field and released to move out on depolarization. Their outward movement must be catalyzed by the voltage sensor to reduce the large thermodynamic barrier to movement of charged amino acid residues across the membrane. The molecular mechanism by which the gating charges are stabilized in the hydrophobic transmembrane environment and the catalytic mechanism through which they are transported across the membrane in response to changes in membrane potential are the subjects of intense research efforts.Progress has been made in determining high-resolution structures of voltage sensors of K_V and Na_V channels in activated states (17–20). However, high-resolution structures of resting and intermediate states of voltage sensors are unknown. The majority of evidence supports a sliding helix model of the voltage-dependent gating in which the gating charge-carrying arginines in S4 are proposed to sequentially form ion pairs with negatively charged residues in S1–S3 segments during activation of the channel (9–11, 21). However, the structural basis for stabilization of the gating charges in the membrane and catalysis of their movement through the hydrophobic membrane environment remain uncertain. Here, we have integrated bioinformatics analysis of Na_V and K_V channel families using the HHPred homology detection server (22–24), high-resolution structural modeling using the Rosetta Membrane (25–27) and Rosetta Symmetry methods (28), the X-ray structures of the K_v1.2-K_v2.1 chimeric channel and NavAb with activated VSDs (19, 20) and the MlotiK1 CNG channel in the resting state (29), and experimental data showing sequential state-dependent interactions between gating charges in S4 and negatively charged residues in S1–S3 (this work and refs. 30–33). Predictions of the resulting voltage-sensing model are confirmed in this work by disulfide-locking studies and mutant cycle analysis of the interactions of hydrophobic residues in the S4 segment. This model reveals structural details of the voltage-dependent conformational changes in the VSD that stabilize and catalyze gating charge movement and are coupled to opening and closing of the intracellular activation gate of the ion-conducting pore.     

From the Cover: Feature Article: The genomic basis of trophic strategy in marine bacteria

Many marine bacteria have evolved to grow optimally at either high (copiotrophic) or low (oligotrophic) nutrient concentrations, enabling different species to colonize distinct trophic habitats in the oceans. Here, we compare the genome sequences of two bacteria, Photobacterium angustum S14 and Sphingopyxis alaskensis RB2256, that serve as useful model organisms for copiotrophic and oligotrophic modes of life and specifically relate the genomic features to trophic strategy for these organisms and define their molecular mechanisms of adaptation. We developed a model for predicting trophic lifestyle from genome sequence data and tested >400,000 proteins representing >500 million nucleotides of sequence data from 126 genome sequences with metagenome data of whole environmental samples. When applied to available oceanic metagenome data (e.g., the Global Ocean Survey data) the model demonstrated that oligotrophs, and not the more readily isolatable copiotrophs, dominate the ocean''s free-living microbial populations. Using our model, it is now possible to define the types of bacteria that specific ocean niches are capable of sustaining.     

From the Cover: Structural differences in amyloid-β fibrils from brains of nondemented elderly individuals and Alzheimer's disease patients

Although amyloid plaques composed of fibrillar amyloid-β (Aβ) assemblies are a diagnostic hallmark of Alzheimer''s disease (AD), quantities of amyloid similar to those in AD patients are observed in brain tissue of some nondemented elderly individuals. The relationship between amyloid deposition and neurodegeneration in AD has, therefore, been unclear. Here, we use solid-state NMR to investigate whether molecular structures of Aβ fibrils from brain tissue of nondemented elderly individuals with high amyloid loads differ from structures of Aβ fibrils from AD tissue. Two-dimensional solid-state NMR spectra of isotopically labeled Aβ fibrils, prepared by seeded growth from frontal lobe tissue extracts, are similar in the two cases but with statistically significant differences in intensity distributions of cross-peak signals. Differences in solid-state NMR data are greater for 42-residue amyloid-β (Aβ42) fibrils than for 40-residue amyloid-β (Aβ40) fibrils. These data suggest that similar sets of fibril polymorphs develop in nondemented elderly individuals and AD patients but with different relative populations on average.

Amyloid plaques in brain tissue, containing fibrils formed by amyloid-β (Aβ) peptides, are one of the diagnostic pathological signatures of Alzheimer''s disease (AD). Clear genetic and biomarker evidence indicates that Aβ is key to AD pathogenesis (1). However, Aβ is present as a diverse population of multimeric assemblies, ranging from soluble oligomers to insoluble fibrils and plaques, and may lead to neurodegeneration by a number of possible mechanisms (2–7).One argument against a direct neurotoxic role for Aβ plaques and fibrils in AD is the fact that plaques are not uncommon in the brains of nondemented elderly people, as shown both by traditional neuropathological studies (8, 9) and by positron emission tomography (10–13). On average, the quantity of amyloid is greater in AD patients (10) and (at least in some studies) increases with decreasing cognitive ability (12, 14, 15) or increasing rate of cognitive decline (16). However, a high amyloid load does not necessarily imply a high degree of neurodegeneration and cognitive impairment (11, 13, 17).A possible counterargument comes from studies of the molecular structures of Aβ fibrils, which show that Aβ peptides form multiple distinct fibril structures, called fibril polymorphs (18–20). Polymorphism has been demonstrated for fibrils formed by both 40-residue amyloid-β (Aβ40) (19, 21–24) and 42-residue amyloid-β (Aβ42) (22, 25–29) peptides, the two main Aβ isoforms. Among people with similar total amyloid loads, variations in neurodegeneration and cognitive impairment may conceivably arise from variations in the relative populations of different fibril polymorphs. As a hypothetical example, if polymorph A was neurotoxic but polymorph B was not, then people whose Aβ peptides happened to form polymorph A would develop AD, while people whose Aβ peptides happened to form polymorph B would remain cognitively normal. In practice, brains may contain a population of different propagating and/or neurotoxic Aβ species, akin to prion quasispecies or “clouds,” and the relative proportions of these and their dynamic interplay may affect clinical phenotype and rates of progression (30).Well-established connections between molecular structural polymorphism and variations in other neurodegenerative diseases lend credence to the hypothesis that Aβ fibril polymorphism plays a role in variations in the characteristics of AD. Distinct strains of prions causing the transmissible spongiform encephalopathies have been shown to involve different molecular structural states of the mammalian prion protein PrP (30–32). Distinct tauopathies involve different polymorphs of tau protein fibrils (33–37). In the case of synucleopathies, α-synuclein has been shown to be capable of forming polymorphic fibrils (38–40) with distinct biological effects (41–43).Experimental support for connections between Aβ polymorphism and variations in characteristics of AD comes from polymorph-dependent fibril toxicities in neuronal cell cultures (19), differences in neuropathology induced in transgenic mice by injection of amyloid-containing extracts from different sources (44–46), differences in conformation and stability with respect to chemical denaturation of Aβ assemblies prepared from brain tissue of rapidly or slowly progressing AD patients (47), and differences in fluorescence emission spectra of structure-sensitive dyes bound to amyloid plaques in tissue from sporadic or familial AD patients (48, 49).Solid-state NMR spectroscopy is a powerful method for investigating fibril polymorphism because even small, localized changes in molecular conformation or structural environment produce measurable changes in ¹³C and ¹⁵N NMR chemical shifts (i.e., in NMR frequencies of individual carbon and nitrogen sites). Full molecular structural models for amyloid fibrils can be developed from large sets of measurements on structurally homogeneous samples (21, 25, 26, 29, 38, 50). Alternatively, simple two-dimensional (2D) solid-state NMR spectra can serve as structural fingerprints, allowing assessments of polymorphism and comparisons between samples from different sources (22, 51).Solid-state NMR requires isotopic labeling and milligram-scale quantities of fibrils, ruling out direct measurements on amyloid fibrils extracted from brain tissue. However, Aβ fibril structures from autopsied brain tissue can be amplified and isotopically labeled by seeded fibril growth, in which fibril fragments (i.e., seeds) in a brain tissue extract are added to a solution of isotopically labeled peptide (21, 22, 52). Labeled “daughter” fibrils that grow from the seeds retain the molecular structures of the “parent” fibrils, as demonstrated for Aβ (19, 21, 24, 53) and other (54, 55) amyloid fibrils. Solid-state NMR measurements on the brain-seeded fibrils then provide information about molecular structures of fibrils that were present in the brain tissue at the time of autopsy. Using this approach, Lu et al. (21) developed a full molecular structure for Aβ40 fibrils derived from one AD patient with an atypical clinical history (patient 1), showed that Aβ40 fibrils from a second patient with a typical AD history (patient 2) were qualitatively different in structure, and showed that the predominant brain-derived Aβ40 polymorph was the same in multiple regions of the cerebral cortex from each patient. Subsequently, Qiang et al. (22) prepared isotopically labeled Aβ40 and Aβ42 fibrils from frontal, occipital, and parietal lobe tissue of 15 patients in three categories, namely typical long-duration Alzheimer''s disease (t-AD), the posterior cortical atrophy variant of Alzheimer''s disease (PCA-AD), and rapidly progressing Alzheimer''s disease (r-AD). Quantitative analyses of 2D solid-state NMR spectra led to the conclusions that Aβ40 fibrils derived from t-AD and PCA-AD tissue were indistinguishable, with both showing the same predominant polymorph; that Aβ40 fibrils derived from r-AD tissue were more structurally heterogeneous (i.e., more polymorphic); and that Aβ42 fibrils derived from all three categories were structurally heterogeneous, with at least two prevalent Aβ42 polymorphs (22).In this paper, we address the question of whether Aβ fibrils that develop in cortical tissue of nondemented elderly individuals with high amyloid loads are structurally distinguishable from fibrils that develop in cortical tissue of AD patients. As described below, quantitative analyses of 2D solid-state NMR spectra of brain-seeded samples indicate statistically significant differences for both Aβ40 and Aβ42 fibrils. Differences in the 2D spectra are subtle, however, indicating that nondemented individuals and AD patients do not develop entirely different Aβ fibril structures. Instead, data and analyses described below suggest overlapping distributions of fibril polymorphs, with different relative populations on average.     

From the Cover: Small molecule blockers of the Alzheimer Aβ calcium channel potently protect neurons from Aβ cytotoxicity

Alzheimer's disease (AD) is a common, chronic neurodegenerative disease that is thought to be caused by the neurotoxic effect of the Amyloid beta peptides (Aβ). We have hypothesized that the intrinsic Aβ calcium channel activity of the oligomeric Aβ polymer may be responsible for the neurotoxic properties of Aβ, and that Aβ channel blockers may be candidate AD therapeutics. As a consequence of a rational search paradigm based on the model structure of the Aβ channel, we have identified two compounds of interest: MRS2481 and an enatiomeric species, MRS2485. These are amphiphilic pyridinium salts that both potently block the Aβ channel and protect neurons from Aβ toxicity. Both block the Aβ channel with similar potency (≈500 nM) and efficacy (100%). However, we find that inhibition by MRS2481 is easily reversible, whereas inhibition by MRS2485 is virtually irreversible. We suggest that both species deserve consideration as candidates for Alzheimer's disease drug discovery.     

Structural basis for GTP-induced dimerization and antiviral function of guanylate-binding proteins

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP5_1–486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP5_1–486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.

Guanylate binding proteins (GBPs) are a family of interferon (IFN)-inducible guanosine triphosphatases (GTPases) that play important roles in innate immunity against diverse intracellular pathogens (1). Many GBPs show activities against bacterial and protozoan pathogens, such as Toxoplasma gondii, Chlamydia trachomatis, Legionella, and Mycobacterium tuberculosis (2). Some of them also have antiviral functions (3). Recently, human GBP5 (hGBP5) was found to restrict HIV-1 by interfering with the processing and incorporation of the viral envelope glycoprotein (Env) (4). A follow-up study revealed that hGBP5 and its paralogue hGBP2 suppress the activity of the virus-dependency factor furin, thereby inhibiting the proteolytic processing of the immature Env precursor gp160 into mature gp120 and gp41 required for virion infectivity (5). Furin is critical for proteolytic cleavage of many viral envelope proteins (6). In support of a key role in innate antiviral immunity, hGBP2 and hGBP5 also restrict other furin-dependent viruses, such as measles, Zika, and highly pathogenic avian influenza A viruses (5).GBPs belong to the dynamin superfamily of large GTPases (7). These are characterized by an N-terminal large GTPase domain (LG domain) and one or more stalk domains (8), usually involved in oligomerization. The stalk domain of GBPs, which is also called C-terminal helical domain (CTHD), comprises the middle domain (MD) and GTPase effector domain (GED). It was proposed that GBPs undergo conformational changes and/or oligomerization upon GTP binding and hydrolysis (9), which may be important for their innate immune functions. Furthermore, GBP1, GBP2, and GBP5 are isoprenylated, and their membrane-binding abilities are modulated by the nucleotide state (10).Despite the importance of this protein family in innate immunity and decades of research, their oligomerization mechanisms remain elusive due to limited structural data (11). The crystal structure of full-length human GBP1 (hGBP1^FL) was determined in its monomeric state (12). The crystal structure of the LG domain alone showed that it is able to form a dimer upon GTP binding (13). Based on these structures, a model of hGBP1^FL in the nucleotide-bound dimeric state was proposed, where the stalk domains protrude to the opposite direction, resulting in an “open” conformation (13). However, the accuracy of this model remains to be tested. Hence, the structures of full-length GBPs in their oligomeric state are in high demand to reveal the detailed molecular mechanisms of GBPs during innate immune responses.Here, we report the crystal structures of hGBP5_1–486 in both its nucleotide-free monomeric state and nucleotide-bound dimeric state, as well as full-length, nucleotide-free human GBP2 (hGBP2^FL). The structures of hGBP5_1–486 and hGBP2^FL are similar to that of hGBP1^FL in the absence of nucleotide. Upon nucleotide binding, however, the stalk domain of hGBP5 undergoes a drastic movement relative to the dimerized LG domain, resulting in a “closed” conformation entirely different from the previously proposed model. Two MD form a hydrophobic interface. Disrupting this interface or mutating the hinge region connecting LG domain and MD, reduces the anti–HIV-1 activity of hGBP2/5, suggesting a crucial role of the closed conformation in their antiviral function. Although the immune functions of the GBP family members are diverse and require specific signals, this dimerization mode is probably shared by all members of the family as revealed by small-angle X-ray scattering (SAXS). On these grounds, we propose a GTP-induced dimerization mechanism of GBPs which lays the foundation to understand the molecular bases of this important innate immune protein family.     

From the Cover: Human autoreactive T cells recognize CD1b and phospholipids

In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αβ T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids.Whereas most studies of the human αβ T-cell response have focused on peptide antigens bound to MHC class I or II molecules, human CD1 proteins represent a parallel system of antigen display that allows T cells to recognize and respond to lipids. Similar to MHC II proteins, human CD1 antigen-presenting molecules (CD1a, CD1b, CD1c, CD1d) are expressed at high density on the surface of professional antigen-presenting cells (APCs), such as myeloid dendritic cells (DCs), Langerhans cells, and B cells (1). CD1 proteins initially capture self-lipids in the endoplasmic reticulum or the secretory pathway, and the cytoplasmic tails of CD1b, CD1c, and CD1d proteins mediate transport to lysosomes for antigen capture at low pH (2). However, unlike MHC proteins, the inner surface of CD1 clefts is lined by hydrophobic, rather than polar or charged amino acids, so there is almost no overlap in the chemical nature of antigens displayed by MHC and CD1 proteins. Thus, the spectrum of natural antigens for T cells is broader than previously thought, creating a situation in which lipids might be developed as the basis for detecting or activating human T cells.In addition to CD1d, humans and most other mammals express CD1a, CD1b, and CD1c proteins, which are known as group 1 CD1 proteins. Prior work using human T-cell clones (3–7) and the recently developed human CD1a, CD1b, and CD1c tetramers (8–10) has demonstrated the existence of mycobacteria-reactive T cells, including polyclonal T cells with stereotyped T-cell receptors (TCRs), known as germline-encoded mycolyl lipid-reactive T cells (11) and LDN5-like T cells (12). At this time, nearly all known foreign antigens presented by CD1b, including mycolic acid, glucose monomycolate, glycerol monomycolate, and sulfoglycolipids, are selectively synthesized by Mycobacterium tuberculosis and related mycobacterial species. Whether or not CD1b functions more broadly in human T-cell responses to other types of bacteria is unknown. To address the breadth of pathogens recognized by CD1b-mediated T cells, we studied human T-cell responses to pathogenic Salmonella, Staphylococcus, and Brucella species. CD1b tetramers have recently been proven to markedly enhance the rate of detection and recovery of CD1b-reactive T cells (8, 11, 12). However, tetramers are not usually used for antigen discovery because they require that homogenous antigen preparations be loaded onto MHC or CD1 proteins. That is, two or more arms of the tetramer must be loaded with the same or similar antigen, which is usually needed to create a multimeric ligand with sufficient avidity to bind TCRs.However, we reasoned that bacterial lipid mixtures could be screened for T-cell response, using higher-order CD1b multimers formed on dextran polymer backbones, which are known as dextramers. This strategy is based on the premise that the higher valence can increase the chance that two or more of the CD1b proteins capture equivalent lipids to create TCR binding epitopes. Whereas mycobacteria express at least 119 classes of lipids defined by Lipid Maps and the MycoMass databases (13), Staphylococcus, Salmonella, and Brucella species produce a much simpler lipid envelope, the content of which is dominated by membrane phospholipids, which are polar and elute in methanol from normal-phase silica columns. Further, prior efforts to discover lipid antigens for CD1 proteins have isolated antigens from methanol eluents of silica columns (4, 14). Therefore, use of methanol eluents from bacteria represented a semitargeted approach that seems to enrich for amphipathic molecules that possess CD1 binding properties. This screening approach succeeded in reproducibly detecting human αβ T cells responding to all three bacterial species, thereby expanding the scope of pathogens recognized by the CD1b system. Surprisingly, studies aimed at solving the chemical structure of the lipid antigens showed that the optimal molecular targets were phospholipids that are synthesized both in mammalian cells and pathogens. Further, recognition of self-lipids by T cells was accompanied by autoreactivity to CD1b expressed on human cells. We show that the molecular basis of self-lipid and foreign-lipid antigen recognition required αβ TCR binding to CD1b–phospholipid complexes, but data pointed away from differences in self-lipid and foreign-lipid structure as the determinant of T-cell response. Instead, these studies showed that CD1b-presented antigens are rare in in the membranes from which CD1b proteins capture self-lipids, but are highly abundant in bacteria.     

From the Cover: Structural adaptations to diverse fighting styles in sexually selected weapons

The shapes of sexually selected weapons differ widely among species, but the drivers of this diversity remain poorly understood. Existing explanations suggest weapon shapes reflect structural adaptations to different fighting styles, yet explicit tests of this hypothesis are lacking. We constructed finite element models of the horns of different rhinoceros beetle species to test whether functional specializations for increased performance under species-specific fighting styles could have contributed to the diversification of weapon form. We find that horns are both stronger and stiffer in response to species-typical fighting loads and that they perform more poorly under atypical fighting loads, which suggests weapons are structurally adapted to meet the functional demands of fighting. Our research establishes a critical link between weapon form and function, revealing one way male–male competition can drive the diversification of animal weapons.Sexually selected traits are renowned for their extreme size and diversity (1, 2). Some sexual traits, such as elaborate feathers in birds of paradise and widowbirds, are used as ornaments to attract choosy females, whereas others, such as giant elk antlers and stag beetle mandibles, are used as weapons in male–male battles over access to females. Numerous empirical (3–9) and theoretical (10–15) studies have shown how female choice can drive the diversification of male ornaments. Surprisingly few studies, however, have examined whether male–male competition drives the diversification of weapons, and the mechanisms responsible for weapon divergence remain largely unexplored (16). As a consequence, although sexually selected weapons are just as diverse as ornaments, it is not clear why this should be so.The most intuitive explanation for weapon diversity is that weapons are adapted to species-specific fighting styles. Specifically, differences either in the way males fight or in where they fight may favor corresponding changes in weapon shape (16). This hypothesis has been explored most thoroughly for the horns and antlers of ungulates (17–20). For example, males in species with short, smooth horns tend to be stabbers; males with robust, curved horns typically ram opponents; and males with long, reaching horns wrestle or fence (18, 19). Although these broad comparative patterns provide evidence that different fighting styles have contributed to the divergence of weapon forms, all the studies are correlative. Explicit tests of the functional performance of weapons in response to forces incurred during fights are still lacking, and no studies have tested whether animal weapons perform better at their own style of fighting than they do at others. Thus, although functional specialization of weapons for diverse styles of fighting remains the most intuitive and widely cited driver of weapon diversity, it has yet to be directly tested for any type of animal weapon.Rhinoceros beetles (Coleoptera: Dynastinae) are ideal for studying weapon diversity for three reasons. First, species vary in the number, size, and shape of their horns, with species wielding long pitchforks, robust pincers, or thin spears, to name just a few of the diverse horn types (16, 21) (Figs. 1 and ​and2).2). Second, horns are used as weapons during combat with rival males over access to females. There is no evidence that females choose males on the basis of the shape or size of their horns (22–25), so horn morphology is expected to reflect differences in how horns are used during fights without conflicting selective pressures from female choice. Third, species fight on a variety of substrates (e.g., on broad tree trunks, on narrow shoots, or inside tunnels) and use their horns in different ways, which may select for qualitatively different fighting structures (16, 23, 24).Open in a separate windowFig. 1.Variation in horn morphology and fighting styles in rhinoceros beetles. (A) Trypoxylus dichotomus males have a long, forked head horn that is used like a pitchfork to lift and twist opponents off tree trunks during fights. (B) Dynastes hercules males have a long head horn and long thoracic horn that are used together similar to pliers to lift, squeeze, and then toss opponents to the ground. (C) Golofa porteri males have a long, slender head horn that is used similar to a fencing sword to both lift opponents off narrow shoots and push them sideways off balance. Vectors represent the typical forces experienced by horns during fights: vertical bending (red), lateral bending (blue), twisting (green). Illustrations by David J. Tuss.Open in a separate windowFig. 2.Horns are stronger under species-specific fighting loads. Von Mises stress distributions and maximum stress values from finite element models of Trypoxylus (A–C), Dynastes (D–F), and Golofa (G–I) horns under vertical bending (A, D, G), lateral bending (B, E, H), and twisting (C, F, I) loads. Typical fighting loads for each species are outlined in gray; atypical fighting loads are not outlined. In all three species, maximum Von Mises stresses in the horn are higher (warmer colors) under atypical loading conditions, indicating a higher likelihood of breaking. Contour plots are scaled to 80 MPa maximum stress. The high stresses at the base of the horn are artifacts from constraining the models and are not included in calculating the maximum stress values in the horn.Here, we perform a functional analysis of rhinoceros beetle horns to test whether horns are structurally suited for diverse fighting styles. Specifically, we compare the mechanical performance of various beetle horn morphologies using finite element analysis, a standard and powerful engineering analysis technique used to predict how complex structures deform, and ultimately fail, in response to applied loads (26). We test whether beetle horns are adapted to meet the functional demands of fighting by constructing finite element models of the head horns of three rhinoceros beetle species and loading the model horns in ways that mimic the forces incurred during both species-typical and species-atypical fights.     

From the Cover: Megafauna and ecosystem function from the Pleistocene to the Anthropocene

Large herbivores and carnivores (the megafauna) have been in a state of decline and extinction since the Late Pleistocene, both on land and more recently in the oceans. Much has been written on the timing and causes of these declines, but only recently has scientific attention focused on the consequences of these declines for ecosystem function. Here, we review progress in our understanding of how megafauna affect ecosystem physical and trophic structure, species composition, biogeochemistry, and climate, drawing on special features of PNAS and Ecography that have been published as a result of an international workshop on this topic held in Oxford in 2014. Insights emerging from this work have consequences for our understanding of changes in biosphere function since the Late Pleistocene and of the functioning of contemporary ecosystems, as well as offering a rationale and framework for scientifically informed restoration of megafaunal function where possible and appropriate.